Isolated chemotactic factor from patients with endometriosis

ABSTRACT

A composition comprising a soluble peptide of about 23 kD to about 29 kD, capable of causing neutrophil and macrophage chemotaxis, and which is substantially similar to chemotactic factor from peritoneal fluid of mammals with minimal or moderate endometriosis.

REFERENCE TO GOVERNMENT GRANTS

This work was supported in part by research grants from the NationalInstitutes of Health, grant numbers HD-20025, HD-06274 and HD-30475. TheUnited States Government may have certain rights in this invention.

This is a continuation of application Ser. No. 08/195,693, filed Feb.16, 1994, now abandoned.

BACKGROUND OF THE INVENTION

The underlying mechanisms indicating an association between minimal tomoderate endometriosis and infertility are not clearly understood.Recent investigations have focused on the presence of an asepticinflammation of the peritoneal cavity, resulting in a distortion of thenormal function of the pelvic organs, as an important contributorymechanism. Other proposed causes for reproductive failure in the lesssevere cases of endometriosis include oligoanovulation, luteal phasedefects, and luteinized unruptured follicle syndrome. However, thesesuggestions need to be clearly substantiated and each one cannot beconsidered the sole cause of infertility in patients with endometriosis.Other factors, which include alterations in the sperm-egg interactionwith possible phagocytosis of the sperm or interference with earlyembryo development, may be taken into consideration when studying theassociated infertility of these patients. The biochemical modificationsthat have been described in endometriosis include an increase in theconcentration of prostaglandins, cytokines and complement components inthe peritoneal fluid and activation of resident macrophages. Halme J, etal., Am J Obstet Gynecol 1983 145:333 and Hill J A, et al., Am J ObstetGynecol 1989 161:861. The activation of leukocytes within the peritonealcavity is evidenced by cytoskeletal rearrangement of the cells and bychanges in the lipid metabolism with activation of protein kinases orrelease of lysosomal enzymes.

The role of peritoneal fluid in the physiologic modifications of theperitoneal cavity of patients who otherwise would seem to have a normalpelvic environment has been studied. Endometriosis without severeanatomic distortion is associated with an increase in the peritonealfluid volume, cell number, and concentration of lysosomic enzymes ascompared with normal fertile controls. The peritoneal fluid arisesprimarily from two different sources: the plasma as a transudate and theovary as an exudate; other sources are tubal fluid; retrogrademenstruation, and secretions from the macrophages in the cavity.However, the exact source for the biochemical modifications observed inendometriosis is not clear.

Normally the peritoneal fluid contains several types of blood cells,with macrophages and lymphocytes being the most abundant; desquamatedendometrial and mesothelial cells are also present. This cellularcomposition is modified in patients with endometriosis. Badaway S. Z.,et al., Fertil Steril 1984 42:704. Several theories exist regarding themechanisms responsible for these alterations, one of which is consideredby the present invention--the presence of a chemotactic stimulus thatwould attract more cells into the peritoneal cavity, or alternatively,activate and induce proliferation of resident macrophages in response tothese unknown factors.

Therefore to study the modifications seen within the pelvic cavity inendometriosis investigators have focused on the initial stages of thedisease in which active endometrial glands and stroma are present andare clearly associated with changes in the peritoneal fluid. Some of themodifications include an increase in the concentrations of cytokines andthe production of various growth factors by the infiltrating leukocytes.The contribution of these factors to the inflammatory changes of theperitoneal fluid is yet to be determined. Fakih H, et al., Fertil Steril1987 47:213 and Halme J, et al., J Clin Endocrinol Metab 1988 66:1044.

The role of the peritoneal fluid of patients with minimal to moderateendometriosis was examined in the present invention as a contributor tothe inflammatory changes observed in the pelvic cavity of thesepatients. Leiva, M., et al., Am J Obstet and Gynecol 1993 168:592. Inthis regard, the chemotactic potential of peritoneal fluid obtained frompatients with minimal to moderate endometriosis was investigated.Responsive cells included neutrophils isolated from peripheral blood orHL60-C15-differentiated neutrophils and macrophages derived from U937cells. These findings were compared with the activity observed in normalpatients or patients undergoing medical treatment for this condition.

These results determine the potential of the invention to satisfy thegreat needs in the art of diagnosis and treatment of endometriosis.Accurate and early methods of detection of endometriosis are needed suchthat methods of treatment may begin promptly.

SUMMARY OF THE INVENTION

The present invention is directed to a composition comprising a solublepeptide of about 23 kD to about 29 kD. The soluble peptide size isdetermined by separation on a G-75 SEPHADEX® column followed by G-25SEPHADEX®/Blue SEPHAROSE® column, a second G-75 SEPHADEX® column, a AntiIg column and HPLC purification. An alternative purification schemeresulting in the same size protein having chemotactic activity includesthe steps of G-75 SEPHADEX® column, ethanol precipitation, Anti Igcolumn purification. The soluble peptide is capable of causingneutrophil and macrophage chemotaxis, and is substantially similar tochemotactic factor from peritoneal fluid of mammals with minimal tomoderate endometriosis.

An antibody preparation specific for a chemotactic factor and acomposition comprising a therapeutically effective amount of an antibodyand a pharmaceutically acceptable carrier useful in the treatment ofendometriosis are included in the scope of the present invention.

A method of detecting endometriosis in a mammal comprising obtaining asample of peritoneal fluid from a mammal suspected of havingendometriosis, contacting the sample with an antibody specific forchemotactic factor, forming antibody-antigen complexes comprisingchemotactic factor and an antibody specific for chemotactic factor, anddetecting antibody-antigen complexes is provided for in the presentinvention.

A method of treating a mammal having endometriosis comprising screeningthe mammal suspected of having endometriosis for the presence ofchemotactic factor and contacting the mammal with an antibody tochemotactic factor is embodied by the present invention.

A diagnostic kit comprising an antibody for the detection of achemotactic factor, a means for detecting chemotactic factor, andoptionally containing positive and negative controls and a solid supportwhich is useful in the detection of endometriosis is also included inthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets forth a graph of chemotactic activity of peritoneal fluid toHL60-differentiated neutrophils. Peritoneal fluid from control,endometriosis, and treatment patients was analyzed for chemotacticactivity with 50,000 HL60 cells differentiated to neutrophils.Background was determined with phosphate buffered saline solution.Results are expressed as number of neutrophils per well ±SD.Asterisk=p<0.001; fMLP=N-formyl-L-methionyl-L-leucyl-L-phenylalanine.

FIG. 2 is a graph of chemotactic activity of peritoneal fluid tomacrophages. U937 cells were differentiated to macrophages as describedin Material and methods and used at a concentration of 1×10⁶ cells permilliliter. Results are expressed as number of macrophages that migratedto lower side of membrane per well ±SD. Positive and negative controlsare as described in FIG. 1.fMLP=N-formyl-L-methionyl-L-leucyl-L-phenylalanine; PBS=phosphatebuffered saline solution.

FIGS. 3A-3B exhibit protein profiles of peritoneal fluid from controland endometriosis patients. Equal amounts of total protein were appliedto a G-75 superfine SEPHADEX® column and eluted with buffer. The 280 nmabsorbance of each fraction was determined. FIG. 3A, Protein profile fornormal patients; FIG. 3B, profile for endometriosis. Column wascalibrated with blue dextran of 2,000,000 molecular weight; V_(o)soybean trypsin inhibitor of 20,000 molecular weight; and cytochrome Cof 12,5000 molecular weight.

FIG. 4 displays chemotactic activity of peritoneal fluid afterfractionation in a G-75 SEPHADEX® column. A 30 μl aliquot of eachfraction was assayed in triplicate for chemotactic activity. Hatchedbars=endometriosis samples; Solid bars=control samples. Results areexpressed as number of neutrophils per well ±SD. Value for positivecontrol (N-formyl-L-methionyl-L-leucyl-L-phenylalanine, 10⁻⁶ mol/L) of592±88.8 and background activity phosphate buffered saline solution(PBS) of 112±58 are shown. Asterisk=p<0.001 between control andendometriosis fractions.

FIG. 5 displays sodium dodecyl sulfate polyacrylamide gelelectrophoresis analysis of chemotactic fractions from peritoneal fluidof patients with endometriosis and their equivalents from disease-freecontrols. A total of 20 μg of protein was loaded into each lane.Arrow=protein and of approximately 20 kD present only in endometriosisfractions.

FIG. 6 exhibits a protein profile of peritoneal fluid from endometriosispatients following G-75 SEPHADEX® column purification. The purificationresulted in three peaks: 90-60 kD molecular weight, 45-20 kD molecularweight, and 15-12 kD molecular weight. Equal amounts of total proteinwere applied to a G-75 superfine SEPHADEX® column and eluted withbuffer. The 280 nm absorbance of each fraction was determined.

FIG. 7 displays a protein profile of peritoneal fluid from endometriosispatients following G-25 SEPHADEX®/Blue SEPHAROSE® column purification.The purification resulted in two peaks: 90-60 kD molecular weight, 45-20kD molecular weight Equal amounts of total protein were applied to aG-25 SEPHADEX®/blue SEPHAROSE® column and eluted with buffer. The 280 nmabsorbance of each fraction was determined.

FIG. 8 is a graph of chemotactic activity of endometriosis andnon-endometriosis peritoneal fluid.N-formyl-L-methionyl-L-leucyl-L-phenylalanine, FMLP, is a knownchemotactic positive control, ENDO--stage I-II patients, III--stage IIIpatients, IV--stage IV patients, NO--patients without endometriosis, andT-10--10 mmol/L Tris pH 7.4, negative control.

FIG. 9 is a graph of chemotactic activity of G-75 SEPHADEX® columnfractions. The box in the upper left corner of the graph sets forth thefractions numbers of the G-75 SEPHADEX® column purification tested whichcorrespond to the fractions in FIG. 6.N-formyl-L-methionyl-L-leucyl-L-phenylalanine, FMLP, is a knownchemotactic positive control and T-10, 10 mmol/L Tris pH 7.4, is anegative control. The numbers above each bar represent the mean.

FIG. 10 is a graph of chemotactic activity of G-25 SEPHADEX®/BlueSEPHAROSE® column fractions. The box in the upper left corner of thegraph sets forth the fractions numbers of the G-25 SEPHADEX®/BlueSEPHAROSE ® column purification tested which correspond to the fractionsin FIG. 7. N-formyl-L-methionyl-L-leucyl-L-phenylalanine, FMLP, is aknown chemotactic positive control and T-10, 10 mmol/L Tris pH 7.4, is anegative control. The numbers above each bar represent the mean.

FIG. 11 is a gel stained with comassie blue. Lanes A and B representsamples which have been purified by G-75 SEPHADEX® column, G-25SEPHADEX®/Blue SEPHAROSE®, and G-75 SEPHADEX® columns; Marker representsthe molecular weight marker lane.

FIG. 12 displays the results of HPLC, two of the fractions, measured atabsorbance₂₁₅, were found to be chemotactic.

FIG. 13 displays the results of a comassie stained gel. The sample lanerepresents the G-75, EtOH precipitated, Protein G purified chemotacticfactor, Marker represents the molecular weight marker lane.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a composition comprising a solublepeptide of about 23 kD to about 29 kD, as determined by the purificationprotocols disclosed herein, which is capable of causing neutrophil andmacrophage chemotaxis. The peptide is isolatable from peritoneal fluidof mammals with minimal to moderate endometriosis. The present inventionincludes peptides isolated from sources other than peritoneal fluid ofmammals with minimal to moderate endometriosis which may besubstantially similar to chemotactic factor from peritoneal fluid.

The composition of the present invention is referred to herein as asoluble peptide, chemotactic factor, chemotactic peptide, and the like.The soluble peptide of the present invention is about 15 kD to about 20kD, as purified by G-75 SEPHADEX® column followed by separation on anSDS polyacrylamide gel. The soluble peptide of the present invention ismore preferably about 23 kD to about 29 kD as purified by G-75 SEPHADEX®column, G-25 SEPHADEX®/Blue SEPHAROSE ® column (dextran cross-linkedwith epichlorohydrin/flue beaded agarose), a second G-75 SEPHADEX®column, and a Protein G column, optionally followed by HPLCpurification. An alternative purification scheme which results in achemotactic factor of the present invention includes the steps of G-75column, ethanol precipitation, and a Protein G column.

This peptide was associated with peritoneal fluid of patients withminimal to moderate endometriosis. Accordingly, it is possible tocorrelate the presence of the chemotactic factor with endometriosis.Chemotactic assay results of HL-60-C15 differentiated neutrophils withperitoneal fluid from endometriosis patients revealed 933.2±172 cellsper well. These results are similar to the response observed withisolated peripheral blood neutrophils, 709±13 cells per well. Similarchemotactic activity was also present when the fluid was incubated withmacrophages from U937 cells, 405±72.6 cells per well.

The peptide includes peptides substantially similar, preferablyidentical, to chemotactic factor purified from peritoneal fluid frommammals with minimal to moderate endometriosis. The peptide includes andis not limited to recombinant, synthetic, and naturally purifiedpeptides, and fragments of recombinant, synthetic, and naturallypurified peptides, substantially similar to chemotactic factor purifiedfrom peritoneal fluid from mammals with minimal to moderateendometriosis. Peptides produced by cell culture are also included inthe present invention.

Endometriosis is a complex disease that involves modifications at theanatomic level with the presence of implants present as ectopicendometrium and endometriomas distributed throughout the pelvis; it alsoproduces modifications at the cellular and molecular level mostlyobserved in milder stages of the disease. The present inventiondiscloses an increase in chemotactic activity of peritoneal fluid inpatients with minimal to moderate endometriosis, which may be one of themany contributing mechanisms for the observed aseptic inflammation inthe peritoneal cavity of these patients. The present invention alsodiscloses a chemotactic factor responsible for the increased chemotacticactivity of peritoneal fluid of patients with minimal to moderateendometriosis. Minimal to moderate endometriosis for use in the presentinvention is defined as stage I to stage III endometriosis in accordancewith the American Fertility Society, Revised American Fertility SocietyClassification of Endometriosis. Fertil Steril 1985 43:351, incorporatedherein by reference. Accordingly, minimal to moderate endometriosisincludes mild, or stage II, endometriosis as defined by the AmericanFertility Society.

The cyclic variations in hormone concentrations are necessary for mostof the biochemical modifications observed in the peritoneal fluid inendometriosis. The present invention suggests that an increase inchemotactic activity may be hormonally regulated and further that astate of medically induced anovulation, or suppressed ovulation,suppresses chemotaxis. The suppression of chemotaxis to values evenlower than those observed for fertile controls lends support to thishypothesis. This suppression of chemotactic activity could be consideredan indicator of the success of the treatment. Previous reports indicatea reduction in the pelvic inflammation associated with endometriosisafter treatment with medroxyprogesterone acetate. Haney A. F., et al.,Am J Obstet Gynecol 1988 159:450.

In the present invention, the chemotactic activity of the peritonealfluid is demonstrated with granulocytes and macrophages. Previousreports indicate macrophages respond to chemoattractants in a mannersimilar to that of granulocytes. Kay G. E., et al., Infect Immun 198341:1166. Macrophages are the predominant cell type observed in theperitoneal fluid and in the cavity of patients with endometriosis. Thepresence of chemotactic stimulus that will increase their number mayalso trigger their activation: this activation may result in the releaseof cytokines and cytotoxic factors that may be directly responsible forthe modification in the peritoneal environment.

In accordance with the present invention, a method of detectingendometriosis in a mammal comprising obtaining a sample of peritonealfluid from a mammal suspected of having endometriosis, contacting thesample with an antibody specific for the chemotactic factor, formingantibody-antigen complexes of the chemotactic factor and antibodyspecific for the chemotactic factor, and detecting the antibody-antigencomplexes is also disclosed. The sample may also be obtained from otherbodily fluids such as and not limited to blood, urine, vaginal fluid,and the like.

The present invention is also directed to a method of treating a mammalhaving endometriosis comprising screening a mammal suspected of havingendometriosis for the presence of the chemotactic factor, and contactingthe mammal with an antibody raised to the chemotactic factor.

For purposes of the present invention, mammals include, and are notlimited to the Order Rodentia, such as mice; Order Logomorpha, such asrabbits; more particularly the Order Carnivora, including Felines (cats)and Canines (dogs); and even more particularly the Order Artiodactyla,Bovines (cows) and Suines (pigs); and the Order Perissodactyla,including Equines (horses); and most particularly the Order Primates,Ceboids and Simoids (monkeys) and Anthropoids (humans and apes). Themammals of the most preferred embodiments are humans.

The antibodies useful in the practice of the invention include anymonoclonal antibody, polyclonal antibody, Fab fragment, and chimericantibody, having an affinity to or binding to the naturally occurringchemotactic factor obtained from peritoneal fluid of mammals withminimal to moderate endometriosis. The method of preparing antibodies isknown to those of skill in the art. Particularly, the method of Kohlerand Milstein, Nature 1975 256:495, incorporated herein by reference, maybe used to produce monoclonal antibodies for use in the invention.Polyclonal antibodies, Fab fragments, and chimeric antibodies may beproduced by methods disclosed in Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989) and Harlow and Lane, Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1981), both incorporated herein by reference.

Methods of obtaining peritoneal fluid samples for analysis include anysurgical and nonsurgical technique known in the art. Surgical methodsinclude and are not limited to biopsy, laparoscopy, and laparotomy.Nonsurgical methods include, and are not limited to peritoneal washings,peritoneal brushings, colpotomy, and vaginal ultrasound aspiration.

Methods of detecting chemotactic factor in the peritoneal fluid includeall methods known in the art. These methods include and are not limitedto, immunohistochemistry techniques such as immunoblotting or Westernblotting, immunoperoxidase staining, fluorescein labelingdiaminobenzadine and biotinylation.

The presence of chemotactic factor in peritoneal fluid is correlatedwith minimal to moderate endometriosis. Thus, the methods of diagnosingendometriosis are also within the scope of the invention. As providedherein, screening for chemotactic factor in peritoneal fluid of mammalssuspected of having endometriosis provides a method of diagnosingendometriosis.

The present invention is also directed to a composition comprisingantibody preparations specific for the chemotactic factor, wherein theantibody preparations are in solution or attached to a solid support.The solid support may be any solid support known to those of skill inthe art useful in immunoassays, such as and not limited to SEPHADEX®,protein A coupled agarose beads, and the like. A composition comprisinga therapeutically effective amount of an antibody to the chemotacticfactor and a pharmaceutically acceptable carrier useful in the treatmentof endometriosis is included in the present invention.

The compositions of the present invention may be administered alone ormay generally be administered in admixture with pharmaceuticallyacceptable carrier selected with regard to route of administration. Theantibodies and therapeutical compositions of the present invention maybe administered with a pharmaceutically acceptable carrier, selectedwith regard to the intended route of administration and the standardpharmaceutical practice. Dosages may be set with regard to weight, andclinical condition of the mammal. The proportional ratio of antibodiesto carrier will naturally depend on chemical nature, solubility, andstability, as well as the dosages contemplated. The dosages will alsodepend on such factors as the age and weight of the mammal. The methodof administration may be by any suitable route, including and notlimited to inoculation and injection, for example, intravenous, oral,intraperitoneal, intramuscular, subcutaneous, topical, and by absorptionthrough epithelial or mucocutaneous linings, for example, nasal, oral,vaginal, rectal and gastrointestinal. The selected carrier and mode ofadministration of the present invention may determine the sites in theorganism to which the composition will be delivered. For instance,carriers include and are not limited to topical applications which maybe administered in creams, ointments, gels, oils, emulsions, pastes,lotions and the like. For parenteral administration, sterile aqueoussolutions may be delivered which contain other solutes, for example,sufficient salts, glucose, or dextrose. For oral mode of administration,the present invention may be used in the form of tablets, capsules,lozenges, troches, powders, syrups, elixirs, aqueous solutions andsuspensions, and the like. For oral administration, certain sweeteningand/or flavoring agents may be added, which are known to those of skillin the art.

Diagnostic kits are also within the scope of this invention. Such kitsinclude antibodies for the detection of chemotactic factor; reagents fordetecting the presence of the factor, such as immunoassay type reagents;optionally positive and negative controls; optionally an absorbantdetection device which may contain pre-absorbed antibodies and to whichpatient samples may be applied; and a means for detecting chemotacticfactor when present, such as a dye.

The antibody-antigen complexes comprise the chemotactic factor and anantibody specific for the chemotactic factor. Methods of detecting theantibody-antigen complexes are known to those of ordinary skill in theart and may be found in Harlow, supra.

The present invention is further described in the following examples.These examples are not to be construed as limiting the scope of theappended claims.

EXAMPLE 1 Material and Methods

Peritoneal fluid was collected during surgical procedures, laparoscopyand one laparotomy, in patients at the Hospital of the University ofPennsylvania. Patients were classified into three groups according toanatomic findings during the procedure and clinical history; normalfertile patients who have no history of endometriosis (n=12) 19 to 37years old (31±7 years, mean=SD) patients with minimal to moderateendometriosis, stage I to stage II endometriosis as defined by theAmerican Fertility Society classification, The American FertilitySociety, Fertil Steril, supra, (n=20) 20 to 40 years old (34±7 years),and patients with a previous diagnosis of endometriosis in whom thesurgical procedure was performed as an evaluation of continuing medicaltreatment for the disease (n=8) (32±1 years). All treatments had beeninitiated at least 6 months before the surgical procedure and were stillbeing administered when the laparoscopy was performed. This groupincluded patients receiving medical suppression of endometriosis(danazol, progestational agents (medroxyprogesterone acetate), orcombination, progestins and estrogens, oral contraceptives).

All peritoneal fluid samples were collected by aspiration during theproliferative phase of the menstrual cycle, the stage in a normalmenstrual cycle which is under the influence of estrogen. The aspiratewas obtained from the posterior and anterior cul-de-sac of theperitoneum before any other intervention, to avoid blood contamination.After aspiration into a sterile syringe, samples were centrifuged at2000 g for 10 minutes at 4° C. to remove any possible contaminatingblood; samples with gross blood contamination were discarded. Aftercentrifugation the supernatant was aliquoted and immediately processedin the chemotaxis assay or stored at -70° C. for evaluation at a latertime. The study protocol was approved by the University of PennsylvaniaInvestigational Committee on the Study of Human Beings.

CHEMOTAXIS ASSAY

The chemotaxis assay was performed by means of a 48-well chemotacticchamber (Neuroprobe, Cabin John, Md.) with a 5 μm pore polycarbonatefilter (Nucleopore, Pleasanton, Calif.) as described by Lee Y. H., etal., Endocrinology 1989 125:3022, incorporated herein by reference.Samples, in 30 μl, were applied to the lower chamber and incubated for30 minutes at 37° C. in humidified 5% carbon dioxide and 95% airatmosphere. After incubation, 50 μl of a cell suspension (containing≧50,000 HL-60-C15 differentiated neutrophils) was added to the upperchamber, and the chemotaxis chamber was incubated for another hour underthe same conditions. HL60-C15 cells were cultured in Roswell ParkMemorial Institute (RPMI) media with 10% heat-inactivated fetal calfserum and differentiated to neutrophils by adding 1.25% dimethylsulfoxide (Sigma, St. Louis) for 7 days, as described by Howe R. S., etal., Exp Hematol 1990 18:299, incorporated herein by reference. Thechemotaxis assay was repeated for each patient sample with neutrophilsisolated through a Percoll gradient (Pharmacia, Piscataway, N.J.) fromthe patients' peripheral blood; and U937 cells, a human histiocytic cellline that can be differentiated to macrophages and has been shown todisplay chemotactic activity by Sundstromm C, et al., Int J. Cancer 197617:565 and Kay G. E., et al., supra. Samples testing neutrophils andU937 cells were processed according to the methods set forth above forHL-60-C15 cells with the exception that U937 cells were cultured inRoswell Park Memorial Institute (RPMI) media with 10% serum andstimulated with 1 mmol/L dibutyryl cyclic adenosine 2'-monophosphate(Sigma) for 48 hours to induce their transformation into macrophages.Chemotactic responses with either of the cell types were comparable, asdemonstrated by Howe R. S., et al., supra and Lee Y. H., et al., supra.After 1 hour of incubation the polycarbonate membrane was washed inphosphate-buffered saline solution (Gibco, Grand Island, N.Y.), fixed inmethanol and stained with Wright-Giemsa (Hemacolor, EMI Diagnostics,Biggstown, N.J.). A known chemotactic peptide,N-formyl-L-methionyl-L-leucyl-L-phenylalanine (Sigma) was used aspositive control, and negative controls included phosphate-bufferedsaline solution and 10 mmol/L Tris hydrochloric acid buffer, pH 7.4(T-10). Chemotaxis was assessed by counting the number of neutrophilsthat had migrated to the bottom side of the filter. Chemotactic activityfor each sample was expressed as mean=SD. Statistical significance wascalculated by means of one-way analysis of variance. A ρ value of ≦0.05was considered significant. This experiment was repeated using isolatedperipheral blood neutrophils and U937 cells.

Peritoneal fluid from patients with minimal to moderate endometriosisexhibited chemotactic activity for isolated peripheral bloodneutrophils, HL60-differentiated neutrophils, and U937 cells. Theresults presented in FIG. 1 indicate that the peritoneal fluid frompatients with minimal to moderate endometriosis display chemotacticactivity for HL60 neutrophils approximately equal to that seen with thepositive control (N-formyl-L-methionyl-L-leucyl-L-phenylalanine).Peritoneal fluid chemotactic activity from normal patients wassignificantly different from the endometriosis group, 384.6±140 versus933.2±172 (ρ<0.001), respectively. It is interesting that the fluid ofpatients undergoing treatment demonstrated the lowest activity, 114±45(ρ<0.001 when compared with that of patients with minimal to moderateendometriosis). These results are similar to the response observed withisolated peripheral blood neutrophils, which was 709±13 cells per wellin the endometriosis group, 289±29 for the control group, and 145±20 forthe treatment group. The data in FIG. 2 indicate that this chemotacticactivity was also present when the fluid was incubated with macrophagesfrom U937 cells and that this activity was similar to that observed forneutrophils, which was 405±72.6 for the endometriosis group, 133±56.5for the control group, and 100±2 for the treatment group.

EXAMPLE 2 Chemotactic Factor Partial Purification

To further investigate the nature of the chemotactic activity observedin the peritoneal fluid, pools of six to ten patient samples were passedthrough a G-75 superfine SEPHADEX® column in 10 mmol/L Tris-hydrochloricacid buffer, pH 7.4, which had been previously calibrated with knownprotein standards (blue dextran, bovine serum albumin, ovalbumin,soybean trypsin inhibitor, and cytochrome C, Sigma). Protein profiles ofthe eluant were monitored by a spectrophotometer (absorbance at 280 nm).The major protein peaks were assayed for chemotactic activity. Fractionsdisplaying chemotactic activity and their equivalents from the controlgroup were analyzed by 12% sodium dodecyl sulfate polyacrylamide gelelectrophoresis under standard reducing conditions. Gels were fixed andstained with comassie blue. The protein band in the low molecular weightrange in the endometriosis samples was cut and electroeluted with theIsco electroelutor-concentrator (Isco, Lincoln, Nebr.). Gel pieces fromthe low molecular weight range were placed in a sample cup and elutedwith 3-(cyclohexylamine)-1-propane-sulfonic acid buffer, pH 11, with aconcentration of 5 mmol/L in the inner chamber, and 25 mmol/L in theouter chamber, and 2.5 mmol/L in the sample cup. Electroelution wasperformed for 60 minutes at 8 watts and the eluted protein was testedfor chemotactic activity by means of macrophages differentiated fromU937 cells.

Examination of the protein profile of both endometriosis and fertilenormal patients after separation through a G-75 SEPHADEX® columnrevealed some differences between groups. Both groups displayed asimilar profile, with the majority of the protein content of being ofhigh molecular weight and a small peak of protein in thelow-molecular-weight range. However, samples from patients withendometriosis had an additional protein peak of low molecular weight(FIG. 3A and B). Protein peaks of both groups were tested forchemotaxis; the data in FIG. 1 indicate that the activity of the firstprotein peak that corresponded to proteins of 60 kD and higher molecularweights did not demonstrate any difference. Peritoneal fluid fromendometriosis patients had the majority of its chemotactic activityassociated with the fractions corresponding to the additional proteinpeak of low molecular weight, about 15 kD to about 20 kD. The activitydisplayed by these fractions was significantly different from that ofthe fertile group.

Fractions displaying chemotactic activity from the endometriosis group(fractions 82 and 84) and their equivalents from disease-free patientswere further analyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis under reducing conditions. The gel was stained withcomassie blue, and analysis of these fractions demonstrated a proteinband with an estimated molecular weight of about 20 kD, which waspresent in the samples from patients with minimal to moderateendometriosis (FIG. 5). This protein was electroeluted and tested forchemotactic activity with macrophages, demonstrating a chemotacticresponse three times higher than background (231.1±44 versus 75.7±11).

The results indicate that the peritoneal fluid from patients withendometriosis has increased chemotactic activity for neutrophils andmacrophages. Endometriosis samples have an additional peak of protein inthe range of about 15 kD to about 20 kD after gel-sieving chromatographyon a G-75 SEPHADEX® column, which is responsible for the chemotacticactivity observed in the fluid of these patients. Analysis of thechemotactic fractions by sodium dodecyl sulfate polyacrylamide gelelectrophoresis revealed the presence of a protein band with anestimated molecular weight of about 20 kD only in the endometriosissamples, which is responsible for this activity. The results alsoindicate that patients with medical suppression as treatment forendometriosis have the lowest chemotactic activity.

EXAMPLE 3 Materials and Methods

Peritoneal fluid was collected by aspiration during laparoscopy fromposterior cul de-sac of female patients at the Hospital of theUniversity of Pennsylvania. The study protocol was approved by theUniversity of Pennsylvania Investigational Committee on the Study ofHuman Beings.

Peritoneal fluid was collected from the posterior cul de sac of 84 womenundergoing laparoscopy between February 1992 and June 1993. The patientsranged in age from 15-39. Patients were divided into two main categoriesaccording to the findings at laparoscopy; group I--patients withoutnoted endometriosis (n=39), group II--patients with endometriosis(n=45). Group II was subdivided into three sections according to the AFSclassification of endometriosis, Fertil Steril, supra; stage I-II(n=41), stage III (n=2), stage IV (n=2). The aspirate was collectedprior to any intervention into a sterile syringe, placed on ice andcentrifuged at 2000 g for 10 minutes. The supernatant was aliquoted into3 cc vials and stored at -70° C.

CHEMOTAXIS ASSAY

The chemotaxis assay was performed on each of the samples by aquantitative assay developed by Lee, Y. H., et al., supra, incorporatedherein by reference. A chemotactic chamber having a polycarbonate filter(Neuroprobe, Cabin John, Md.) was used. U937 cells, a macrophage stemcell line, were grown in RPMI (Gibco, Great Island, N.Y.) plus 10%bovine serum albumin (JRH, Lenexa, Kans.) 5% Glutamate (Gibco) and 5%penicillin/ streptomycin, PCN/Strep, (Gibco). Differentiation of theU937 into macrophages was stimulated with the addition of N⁶ 2¹-O-Dibutyryl-adenosine 3¹ 5¹ cyclic monophosphate (Sigma) per 10 cc for48 hours.

The peritoneal fluid from patients with endometriosis, group II, hadsignificantly increased chemotactic activity (mean=148±59) over theperitoneal fluid of patients without endometriosis, group I,(mean=48±26) and over the negative controls (mean=25±15) p<0.001. Thechemotactic activity of group II increased beginning with stage IV(mean=75), stage III (mean=105) and greatest with stage III (mean=153),see FIG. 8. Three of the 39 samples in group I displayed chemotacticactivity comparable with that of group II or positive controls. All ofthe samples in group II displayed chemotactic activity similar topositive controls.

PURIFICATION

Peritoneal fluid from each patient was passed through a G-75 SEPHADEX®(Sigma) column in 10 mm/L tris-hydrochloride buffer (T-l0), pH 7.4,which was previously calibrated with known protein standards. Theprotein profile was monitored by a spectrophotometer (absorbance at 280nm), FIG. 6. The purification resulted in three peaks: about 90-60 kDmolecular weight, about 45-20 kD molecular weight, and about 15-12 kDmolecular weight. The chemotaxis assay was performed on a sample of eachpeak. Peak 2, 45-20 kD, consistently displayed chemotactic activity.

Chemotactic activity of the G-75 SEPHADEX® column fractions is displayedin FIG. 9. The fraction numbers, displayed in a legend in the leftcorner of FIG. 9 correspond to the fraction numbers in FIG. 6. The meanfor FMLP was 150, Fraction 67 provided the highest chemotactic activityof the samples (mean=180), followed by Fraction 95 (mean=85), Fraction20 (mean=45), and the negative control, T-10 and fraction 110 (mean=30).

Peak 2 samples of three patients (stage I-II) were pooled andconcentrated by a speed vacuum and analyzed on a G-25 SEPHADEX®/BlueSEPHAROSE® (Sigma) column in T-10. 2 Molar NaCl (in a volume equivalentto 80% of the sample volume) was added to fractions collected after thebed volume to reveal one chemotactic peak, FIG. 7. This peak providedthe chemotactic protein of about 23 to about 29 kD.

Another chemotactic assay was performed on the fractions identified inFIG. 7. Chemotactic activity of these fractions is set forth in FIG. 10.The fraction numbers, displayed in a legend in the left corner of FIG.10 correspond to the fraction numbers in FIG. 7. The mean for FMLP was150, Fraction 60 provided the highest chemotactic activity of thesamples (mean=175), followed by Fraction 15 (mean=45), Fractions 42, 80,and the negative control, T-10 (mean=30).

Concentrated samples of the chemotactic factor, resulting after G-75SEPHADEX® and samples after G-25 SEPHADEX®/Blue SEPHAROSE® columnpurification, were run on a 12% SDS--polyacrylamide gel electrophoresisunder reducing conditions.

For SDS PAGE, to each sample was added 5 μl of 0.1% bromophenol blue(Na⁺ salt) and 2X final sample buffer (FSB) in an amount equal to thevolume of the sample. 2X FSB is prepared as follows:

    ______________________________________    water                   0.5   ml    Tris HCl (0.5M, pH 6.8) 2.5   ml    glycerol                2.0   ml    SDS (10%)               4.0   ml    β-mercaptoethanol* 1.0   ml    ______________________________________     *for nonreducing gel, omit β-mercaptoethanol

Standards contained 20 μl standard:20 μl 2X FSB. Each sample was a totalvolume of 30 μl. Samples and standards were vortexed, heated at 95° C.for 2 minutes. The SDS gels were then stained with comassie blue.

The G-25 SEPHAREX/Blue SEPHAROSE® fraction was run on a second G-75SEPHADEX® column. FIG. 11 reveals the results of the G-25 SEPHADEX/BlueSEPHAROSE® fraction run on a second G-75 SEPHADEX® column. Comassie bluestaining revealed two bands, about 45 to about 55 kD and about 28 toabout 29 kD. The concentrated samples were then analyzed by a Protein Gcolumn and HPLC. The HPLC results revealed two fractions havingchemotactic activity, FIG. 12.

Concentrated samples of the chemotactic fraction from G-25 SEPHADEX/blueSEPHAROSE® columns were also analyzed on a G-75 SEPHADEX® column. Theprotein peak from this column, measured by absorbance at 280 nm, was runon a 12% SDS gel and stained with comassie blue.

Chromatography, electrophoresis, and chemotaxis assay, set forth above,verified that the 23-29 kD band identified as peak 2 in FIG. 7,contained the chemotactic factor.

EXAMPLE 4

Using the samples obtained for Example 3 above, G-75 SEPHADEX® columnpurification was performed as described above and the samples wereprecipitated in ethanol. All equipment and 100ETOH were placed in -20°C. to chill. Two concentrated samples of the chemotactic fraction fromthe G-75 SEPHADEX® column were placed in a chilled beaker. Cold ETOH wasslowly added to the samples until maximum precipitation was noted at=80% of sample volume. The sample was then centrifuged at 2000 g for 10minutes at 10° C. The supernatant and pellet were separated and thepellet was resuspended in T-10 at pH 7.4.

The chemotaxis assay was performed on the supernatant and on the pellet.Positive chemotaxis noted only in the reconstituted pellet portion, asset forth in Table 1.

                  TABLE 1    ______________________________________           Sample  Mean    ______________________________________           T-10    25           G-75    160           Supernatant                   50           Pellet  110           FMLP    175    ______________________________________

The ethanol precipitated protein in T-10 was then run over a Protein Gcolumn (Spectra Gel, Spectrum) and all fractions collected. The sampleswere concentrated and remained chemotactic, as set forth in Table 2.

                  TABLE 2    ______________________________________            Sample Mean    ______________________________________            T-10   30            G-75   185            After  170            Protein G            column            FLMP   195    ______________________________________

The concentrated sample was run on a 12% SDS gel and stained withcomassie blue. Three bands resulted from the G-75, ethanol precipitated,Protein G purified protein, about 42 kD, about 27 kD, and about 16 kD,see FIG. 13.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

What is claimed:
 1. A purified soluble peptide of about 27 kD asdetermined by purification on a.) a column of dextran cross-linked withepichlorohydrin, b.) a column of dextran cross-linked withepichlorohydrin/blue beaded agarose, c.) a column of dextrancross-linked with epichlorohydrin, d.) an Anti Ig column, and e.) SDSpolyacrylamide gel electrophoresis, said peptide isolated fromperitoneal fluid of mammals with minimal to moderate endometriosis, saidpeptide having chemotactic activity to neutrophils and macrophages.
 2. Apurified soluble peptide of about 27 kD, having chemotactic activity toneutrophils and macrophages, isolated from peritoneal fluid of mammalshaving minimal to moderate endometriosis, as determined by purificationby:a) obtaining a sample of body fluid suspected of containing saidpeptide; b) purifying said sample on a column of dextran cross-linkedwith epichlorohydrin; c) pooling the 50-20 kD molecular weight fractionsthereby forming a 50-20 kD pooled sample; d) purifying said 50-20 kDpooled sample on a column of dextran cross-linked withepichlorohydrin/blue beaded agarose, thereby obtaining a second pooledsample; e) purifying said second pooled sample on a second column ofdextran cross-linked with epichlorohydrin, thereby obtaining a thirdpooled sample; and f) purifying said third pooled sample on an Anti Igcolumn to obtain said soluble peptide.
 3. A purified soluble peptide ofabout 27 kD, said peptide having chemotactic activity againstneutrophils and macrophages and said peptide isolated from peritonealfluid of mammals with minimal to moderate endometriosis, as determinedby purification by:a) obtaining a sample of body fluid suspected ofcontaining said peptide; b) purifying said sample on a column of dextrancross-linked with epichlorohydrin; c) pooling the 50-20 kD molecularweight fractions, thereby forming a pooled sample; d) ethanolprecipitating said pooled sample; and e) purifying said pooled sample onan Anti Ig column thereby producing a peptide of about 27 kD molecularweight.